HADHs as Modifiers of the p21 Pathway and Methods of Use

ABSTRACT

Human HADH genes are identified as modulators of the p21 pathway, and thus are therapeutic targets for disorders associated with defective p21 function. Methods for identifying modulators of p21, comprising screening for agents that modulate the activity of HADH are provided.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 11/103,068, filed Apr. 11, 2005, which is a continuation applicationof U.S. application Ser. No. 11/103,035, filed Apr. 11, 2005, which is adivisional application of U.S. application Ser. No. 10/192,353, filedJul. 10, 2002, issued as U.S. Pat. No. 6,927,022, which claims priorityto U.S. Provisional Application Ser. No. 60/357,452, filed Feb. 15,2002, U.S. Provisional Application Ser. No. 60/328,491, filed Oct. 10,2001, and U.S. Provisional Application Ser. No. 60/305,017, filed Jul.12, 2001, all of which applications are hereby incorporated by referencein their entireties.

BACKGROUND OF THE INVENTION

The p21/CDKN1/WAF1/CIP1 protein (El-Deiry, W. S.; et al. Cell 75:817-825, 1993; Harper, J. W.; et al. Cell 75: 805-816, 1993; Huppi, Ketal. Oncogene 9: 30173020, 1994) is a cell cycle control protein thatinhibits cyclin-kinase activity, is tightly regulated at thetranscriptional level by p21, and mediates p21 suppression of tumor cellgrowth. Along with p21, p21 appears to be essential for maintaining theG2 checkpoint in human cells (Bunz, F.; Dutriaux, A.; et al. Science282:1497-1501, 1998). Sequences of P21 are well-conserved throughoutevolution, and have been identified in species as diverse as human(Genbank Identifier 13643057), Drosophila melanogaster (GI# 1684911),Caenorhabditis el egans (GI#4966283), and yeast (GI#2656016).

The hydrolytic dehalogenases catalyse a nucleophilic displacementreaction, with water as the sole co-substrate. They are divided intohaloalkane dehalogenases and haloacid dehalogenases (HAD). HADs belongto a large superfamily of hydrolases with diverse substrate specificity,which also includes epoxide hydrolases, phosphoglycolate phosphatases,histidinol phosphate phosphatases, nitrophenyl phosphatases and numerousputative proteins. The epoxide hydrolases (EH) add water to epoxides,forming the corresponding diol. HADH (C20orf147) is a member of thehaloacid dehalogenase or epoxide hydrolase family.

The ability to manipulate the genomes of model organisms such asDrosophila provides, a powerful means to analyze biochemical processesthat, due to significant evolutionary conservation, has direct relevanceto more complex vertebrate organisms. Due to a high level of gene andpathway conservation, the strong similarity of cellular processes, andthe functional conservation of genes between these model organisms andmammals, identification of the involvement of novel genes in particularpathways and their functions in such model organisms can directlycontribute to the understanding of the correlative pathways and methodsof modulating them in mammals (see, for example, Mechler B M et al.,1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74;Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin GM. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev5: 4450; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284). Forexample, a genetic screen can be carried out in an invertebrate modelorganism having underexpression (e.g. knockout) or overexpression of agene (referred to as a “genetic entry point”) that yields a visiblephenotype. Additional genes are mutated in a random or targeted manner.When a gene mutation changes the original phenotype caused by themutation in the genetic entry point, the gene is identified as a“modifier” involved in the same or overlapping pathway as the geneticentry point. When the genetic entry point is an ortholog of a human geneimplicated in a disease pathway, such as p21, modifier genes can beidentified that may be attractive candidate targets for noveltherapeutics.

All references cited herein, including sequence information inreferenced Genbank identifier numbers and website references, areincorporated herein in their entireties.

SUMMARY OF THE INVENTION

We have discovered genes that modify the p21 pathway in Drosophila, andidentified their human orthologs, hereinafter referred to as HADH. Theinvention provides methods for utilizing these p21 modifier genes andpolypeptides to identify candidate therapeutic agents that can be usedin the treatment of disorders associated with defective p21 function.Preferred HADH-modulating agents specifically bind to HADH polypeptidesand restore p21 function. Other preferred HADH-modulating agents arenucleic acid modulators such as antisense oligomers and RNAi thatrepress HADH gene expression or product activity by, for example,binding to and inhibiting the respective nucleic acid (i.e. DNA ormRNA).

HADH-specific modulating agents may be evaluated by any convenient invitro or in vivo assay for molecular interaction with an HADHpolypeptide or nucleic acid. In one embodiment, candidate p21 modulatingagents are tested with an assay system comprising a HADH polypeptide ornucleic acid. Candidate agents that produce a change in the activity ofthe assay system relative to controls are identified as candidate p21modulating agents. The assay system may be cell-based or cell-free.HADHmodulating agents include HADH related proteins (e.g. dominantnegative mutants, and biotherapeutics); HADH-specific antibodies;HADH-specific antisense oligomers and other nucleic acid modulators; andchemical agents that specifically bind HADH or compete with HADH bindingtarget. In one specific embodiment, a small molecule modulator isidentified using a binding assay. In specific embodiments, the screeningassay system is selected from an apoptosis assay, a cell proliferationassay, an angiogenesis assay, and a hypoxic induction assay.

In another embodiment, candidate p21 pathway modulating agents arefurther tested using a second assay system that detects changes in thep21 pathway, such as angiogenic, apoptotic, or cell proliferationchanges produced by the originally identified candidate agent or anagent derived from the original agent. The second assay system may usecultured cells or non-human animals. In specific embodiments, thesecondary assay system uses non-human animals, including animalspredetermined to have a disease or disorder implicating the p21 pathway,such as anangiogenic, apoptotic, or cell proliferation disorder (e.g.cancer).

The invention further provides methods for modulating the p21 pathway ina mammalian cell by contacting the mammalian cell with an agent thatspecifically binds a HADH polypeptide or nucleic acid. The agent may bea small molecule modulator, a nucleic acid modulator, or an antibody andmay be administered to a mammalian animal predetermined to have apathology associated the p21 pathway.

DETAILED DESCRIPTION OF THE INVENTION

An overexpression screen was carried out in Drosophila to identify genesthat interact with the cyclin dependent kinase inhibitor, p21 (Bourne HR, et al., Nature (1990) 348(6297):125-132; Marshall C J, Trends Genet(1991) 7(3):91-95). Expression of the p21 gene in the eye causesdeterioration of normal eye morphology. The CG15771 gene was identifiedas a modifier of the p21 pathway. Accordingly, vertebrate orthologs ofthese modifiers, and preferably the human orthologs, HADH genes (i.e.,nucleic acids and polypeptides) are attractive drug targets for thetreatment of pathologies associated with a defective p21 signalingpathway, such as cancer.

In vitro and in vivo methods of assessing HADH function are providedherein. Modulation of the HADH or their respective binding partners isuseful for understanding the association of the p21 pathway and itsmembers in normal and disease conditions and for developing diagnosticsand therapeutic modalities for p21 related pathologies. HADH-modulatingagents that act by inhibiting or enhancing HADH expression, directly orindirectly, for example, by affecting an HADH function such as enzymatic(e.g., catalytic) or binding activity, can be identified using methodsprovided herein. HADH modulating agents are useful in diagnosis, therapyand pharmaceutical development.

Nucleic Acids and Polypeptides of the Invention

Sequences related to HADH nucleic acids and polypeptides that can beused in the invention are disclosed in Genbank (referenced by Genbankidentifier (GI) number) as GI#s 11968366 (SEQ ID NO:1), 18490373 (SEQ IDNO:2), and 20405373 (SEQ ID NO:3) for nucleic acid, and GI# 4902680 (SEQID NO:4) polypeptides. Additionally, polypeptide sequence of SEQ ID NO:5is a translation of SEQ ID NO:2, and can be used in the invention.

HADHs are hydrolase proteins with hydrolase domains. The term “HADHpolypeptide” refers to a full-length HADH protein or a functionallyactive fragment or derivative thereof A “functionally active” HADHfragment or derivative exhibits one or more functional activitiesassociated with a full-length, wild-type HADH protein, such as antigenicor immunogenic activity, enzymatic activity, ability to bind naturalcellular substrates, etc. The functional activity of HADH proteins,derivatives and fragments can be assayed by various methods known to oneskilled in the art (Current Protocols in Protein Science (1998) Coliganet al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as furtherdiscussed below. For purposes herein, functionally active fragments alsoinclude those fragments that comprise one or more structural domains ofan HADH, such as a hydrolase domain or a binding domain. Protein domainscan be identified using the PFAM program (Bateman A., et al., NucleicAcids Res, 1999,27:260-2; http://pfam.wustl.edu). For example, thehydrolase domain of HADH from GI# 4902680 (SEQ ID NO:4) is located atapproximately amino acid residues 9-212 (PFAM 00702). Methods forobtaining HADH polypeptides are also further described below. In someembodiments, preferred fragments are functionally active,domain-containing fragments comprising at least 25 contiguous aminoacids, preferably at least 50, more preferably 75, and most preferablyat least 100 contiguous amino acids of SEQ ID NOs:4 or 5 (an HADH). Infurther preferred embodiments, the fragment comprises the entirehydrolase (functionally active) domain.

The term “HADH nucleic acid” refers to a DNA or RNA molecule thatencodes a HADH polypeptide. Preferably, the HADH polypeptide or nucleicacid or fragment thereof is from a human, but can also be an ortholog,or derivative thereof with at least 70% sequence identity, preferably atleast 80%, more preferably 85%, still more preferably 90%, and mostpreferably at least 95% sequence identity with HADH. Normally, orthologsin different species retain the same function, due to presence of one ormore protein motifs and/or 3-dimensional structures. Orthologs aregenerally identified by sequence homology analysis, such as BLASTanalysis, usually using protein bait sequences. Sequences are assignedas a potential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research (2000) 10:1204-1210). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al, 1994, NucleicAcids Res 22:4673-4680) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. In evolution, when a gene duplication event followsspeciation, a single gene in one species, such as Drosophila, maycorrespond to multiple genes (paralogs) in another, such as human. Asused herein, the term “orthologs” encompasses paralogs. As used herein,“percent (%) sequence identity” with respect to a subject sequence, or aspecified portion of a subject sequence, is defined as the percentage ofnucleotides or amino acids in the candidate derivative sequenceidentical with the nucleotides or amino acids in the subject sequence(or specified portion thereof), after aligning the sequences andintroducing gaps, if necessary to achieve the maximum percent sequenceidentity, as generated by the program WU-BLAST-2.0a19 (Altschul et al.,J. Mol. Biol. (1997) 215:403-410;http://blast.wustl.edu/blast/README.html) with all the search parametersset to default values. The HSP S and HSP S2 parameters are dynamicvalues and are established by the program itself depending upon thecomposition of the particular sequence and composition of the particulardatabase against which the sequence of interest is being searched. A %identity value is determined by the number of matching identicalnucleotides or amino acids divided by the sequence length for which thepercent identity is being reported. “Percent (%) amino acid sequencesimilarity” is determined by doing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation.

A conservative amino acid substitution is one in which an amino acid issubstituted for another amino acid having similar properties such thatthe folding or activity of the protein is not significantly affected.Aromatic amino acids that can be substituted for each other arephenylalanine, tryptophan, and tyrosine; interchangeable hydrophobicamino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Alternatively, an alignment for nucleic acid sequences is provided bythe local homology algorithm of Smith and Waterman (Smith and Waterman,1981, Advances in Applied Mathematics 2:482-489; database: EuropeanBioinformatics Institute http://vvww.ebi.ac.uk/MPsrch/; Smith andWaterman, 1981, J. of Molec. Biol., 147:195197; Nicholas et al., 1998,“A Tutorial on Searching Sequence Databases and Sequence ScoringMethods” (www.psc.edu) and references cited therein; W. R. Pearson,1991, Genomics 11:635-650). This algorithm can be applied to amino acidsequences by using the scoring matrix developed by Dayhoff (Dayhoff:Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res.14(6):6745-6763). The Smith-Waterman algorithm may be employed wheredefault parameters are used for scoring (for example, gap open penaltyof 12, gap extension penalty of two). From the data generated, the“Match” value reflects “sequence identity.”

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNOs:1, 2, or 3. The stringency of hybridization can be controlled bytemperature, ionic strength, pH, and the presence of denaturing agentssuch as formamide during hybridization and washing. Conditions routinelyused are set out in readily available procedure texts (e.g., CurrentProtocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons,Publishers (1994); Sambrook et al., Molecular Cloning, Cold SpringHarbor (1989)). In some embodiments, a nucleic acid molecule of theinvention is capable of hybridizing to a nucleic acid moleculecontaining the nucleotide sequence of any one of SEQ ID NOs:1, 2, or 3under stringent hybridization conditions that comprise: prehybridizationof filters containing nucleic acid for 8 hours to overnight at 65° C. ina solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 MNaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodiumpyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20hours at 65° C. in a solution containing 6×SSC, 1×Denhardt's solution,100 pg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing offilters at 65° C. for 1 h in a solution containing 0.2×SSC and 0.1% SDS(sodium dodecyl sulfate).

In other embodiments, moderately stringent hybridization conditions areused that comprise: pretreatment of filters containing nucleic acid for6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mMTris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mMEDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 1001 tg/ml salmon sperm DNA,and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hourat 55° C. in a solution containing 2×SSC and 0.1% SDS.

Alternatively, low stringency conditions can be used that comprise:incubation for 8 hours to overnight at 37° C. in a solution comprising20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmonsperm DNA; hybridization in the same buffer for 18 to 20 hours; andwashing of filters in 1×SSC at about 37° C. for 1 hour.

Isolation, Production, Expression, and Mis-Expression of HADH NucleicAcids and Polypeptides

HADH nucleic acids and polypeptides, useful for identifying and testingagents that modulate HADH function and for other applications related tothe involvement of HADH in the p21 pathway. HADH nucleic acids andderivatives and orthologs thereof may be obtained using any availablemethod. For instance, techniques for isolating cDNA or genomic DNAsequences of interest by screening DNA libraries or by using polymerasechain reaction (PCR) are well known in the art. In general, theparticular use for the protein will dictate the particulars ofexpression, production, and purification methods. For instance,production of proteins for use in screening for modulating agents mayrequire methods that preserve specific biological activities of theseproteins, whereas production of proteins for antibody generation mayrequire structural integrity of particular epitopes. Expression ofproteins to be purified for screening or antibody production may requirethe addition of specific tags (e.g., generation of fusion proteins).Overexpression of an HADH protein for assays used to assess HADHfunction, such as involvement in cell cycle regulation or hypoxicresponse, may require expression in eukaryotic cell lines capable ofthese cellular activities.

Techniques for the expression, production, and purification of proteinsare well known in the art; any suitable means therefore may be used(e.g., Higgins S J and Hames B D (eds.) Protein Expression: A PracticalApproach, Oxford University Press Inc., New York 1999; Stanbury P F etal., Principles of Fermentation Technology, 2^(11d) edition, ElsevierScience, New York, 1995; Doonan S (ed.) Protein Purification Protocols,Humana Press, New Jersey, 1996; Coligan J E et al, Current Protocols inProtein Science (eds.), 1999, John Wiley & Sons, New York). Inparticular embodiments, recombinant HADH is expressed in a cell lineknown to have defective p21 function such as HCT116 colon cancer cellsavailable from American Type Culture Collection (ATCC), Manassas, Va.).The recombinant cells are used in cell-based screening assay systems ofthe invention, as described further below.

The nucleotide sequence encoding an HADH polypeptide can be insertedinto any appropriate expression vector. The necessary transcriptionaland translational signals, including promoter/enhancer element, canderive from the native HADH gene and/or its flanking regions or can beheterologous. A variety of host-vector expression systems may beutilized, such as mammalian cell systems infected with virus (e.g.vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g. baculovirus); microorganisms such as yeast containing yeastvectors, or bacteria transformed with bacteriophage, plasmid, or cosmidDNA. A host cell strain that modulates the expression of, modifies,and/or specifically processes the gene product may be used.

To detect expression of the HADH gene product, the expression vector cancomprise a promoter operably linked to an HADH gene nucleic acid, one ormore origins—of replication, and, one or more selectable markers (e.g.thymidine kinase activity, resistance to antibiotics, etc.).Alternatively, recombinant expression vectors can be identified byassaying for the expression of the HADH gene product based on thephysical or functional properties of the HADH protein in in vitro assaysystems (e.g. immunoassays).

The HADH protein, fragment, or derivative may be optionally expressed asa fusion, or chimeric protein product (i.e. it is joined via a peptidebond to a heterologous protein sequence of a different protein), forexample to facilitate purification or detection. A chimeric product canbe made by ligating the appropriate nucleic acid sequences encoding thedesired amino acid sequences to each other using standard methods andexpressing the chimeric product. A chimeric product may also be made byprotein synthetic techniques, e.g. by use of a peptide synthesizer(Hunkapiller et al., Nature (1984) 310:105-111).

Once a recombinant cell that expresses the HADH gene sequence isidentified, the gene product can be isolated and purified using standardmethods (e.g. ion exchange, affinity, and gel exclusion chromatography;centrifugation; differential solubility; electrophoresis, citepurification reference). Alternatively, native HADH proteins can bepurified from natural sources, by standard methods (e.g. immunoaffinitypurification). Once a protein is obtained, it may be quantified and itsactivity measured by appropriate methods, such as immunoassay, bioassay,or other measurements of physical properties, such as crystallography.

The methods of this invention may also use cells that have beenengineered for altered expression (mis-expression) of HADH or othergenes associated with the p21 pathway. As used herein, mis-expressionencompasses ectopic expression, over-expression, under-expression, andnon-expression (e.g. by gene knock-out or blocking expression that wouldotherwise normally occur).

Genetically Modified Animals

Animal models that have been genetically modified to alter HADHexpression may be used in in vivo assays to test for activity of acandidate p21 modulating agent, or to further assess the role of HADH ina p21 pathway process such as apoptosis or cell proliferation.Preferably, the altered HADH expression results in a detectablephenotype, such as decreased or increased levels of cell proliferation,angiogenesis, or apoptosis compared to control animals having normalHADH expression. The genetically modified animal may additionally havealtered p21 expression (e.g. p21 knockout). Preferred geneticallymodified animals are mammals such as primates, rodents (preferablymice), cows, horses, goats, sheep, pigs, dogs and cats. Preferrednon-mammalian species include zebrafish, C. elegans, and Drosophila.Preferred genetically modified animals are transgenic animals having aheterologous nucleic acid sequence present as an extrachromosomalelement in a portion of its cells, i.e. mosaic animals (see, forexample, techniques described by Jakobovits, 1994, Curr. Biol.4:761763.) or stably integrated into its germ line DNA (i.e., in thegenomic sequence of most or all of its cells). Heterologous nucleic acidis introduced into the germ line of such transgenic animals by geneticmanipulation of, for example, embryos or embryonic stem cells of thehost animal.

Methods of making transgenic animals are well-known in the art (fortransgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82:4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Lederet al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B.,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No.,4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin andSpradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; fortransgenic insects see Berghammer A. J. et al., A Universal Marker forTransgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafishsee Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-3830); for microinjection procedures for fish, amphibian eggsand birds see Houdebine and Chourrout, Experientia (1991) 47:897-905;for transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and forculturing of embryonic stem (ES) cells and the subsequent production oftransgenic animals by the introduction of DNA into ES cells usingmethods such as electroporation, calcium phosphate/DNA precipitation anddirect injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, APractical Approach, E. J. Robertson, ed., IRL Press (1987)). Clones ofthe nonhuman transgenic animals can be produced according to availablemethods (see Wilmut, I. et al. (1997) Nature. 385:810-813; and PCTInternational Publication Nos. WO 97/07668 and WO 97/07669).

In one embodiment, the transgenic animal is a “knock-out” animal havinga heterozygous or homozygous alteration in the sequence of an endogenousHADH gene that results in a decrease of HADH function, preferably suchthat HADH expression is undetectable or insignificant. Knock-out animalsare typically generated by homologous recombination with a vectorcomprising a transgene having at least a portion of the gene to beknocked out. Typically a deletion, addition or substitution has beenintroduced into the transgene to functionally disrupt it. The transgenecan be a human gene (e.g., from a human genomic clone) but morepreferably is an ortholog of the human gene derived from the transgenichost species. For example, a mouse HADH gene is used to construct ahomologous recombination vector suitable for altering an endogenous HADHgene in the mouse genome. Detailed methodologies for homologousrecombination in mice are available (see Capecchi, Science (1989)244:1288-1292; Joyner et al., Nature (1989) 338:153-156). Procedures forthe production of non-rodent transgenic mammals and other animals arealso available (Houdebine and Chourrout, supra; Pursel et al., Science(1989) 244:1281-1288; Simms et al., Bio/Technology (1988) 6:179-183). Ina preferred embodiment, knock-out animals, such as mice harboring aknockout of a specific gene, may be used to produce antibodies againstthe human counterpart of the gene that has been knocked out (Claesson MH et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995)J Biol Chem. 270:8397-400).

In another embodiment, the transgenic animal is a “knock-in” animalhaving an alteration in its genome that results in altered expression(e.g., increased (including ectopic) or decreased expression) of theHADH gene, e.g., by introduction of additional copies of HADH, or byoperatively inserting a regulatory sequence that provides for alteredexpression of an endogenous copy of the HADH gene. Such regulatorysequences include inducible, tissue-specific, and constitutive promotersand enhancer elements. The knock-in can be homozygous or heterozygous.

Transgenic nonhuman animals can also be produced that contain selectedsystems allowing for regulated expression of the transgene. One exampleof such a system that may be produced is the cre/loxP recombinase systemof bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat.No. 4,959,317). If a cre/loxP recombinase • system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein are required. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected protein and the other containing a transgeneencoding a recombinase. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferredembodiment, both Cre-LoxP and Flp-Frt are used in the same system toregulate expression of the transgene, and for sequential deletion ofvector sequences in the same cell (Sun X et al (2000) Nat Genet25:83-6).

The genetically modified animals can be used in genetic studies tofurther elucidate the p21 pathway, as animal models of disease anddisorders implicating defective p21 function, and for in vivo testing ofcandidate therapeutic agents, such as those identified in screensdescribed below. The candidate therapeutic agents are administered to agenetically modified animal having altered HADH function and phenotypicchanges are compared with appropriate control animals such asgenetically modified animals that receive placebo treatment, and/oranimals with unaltered HADH expression that receive candidatetherapeutic agent.

In addition to the above-described genetically modified animals havingaltered HADH function, animal models having defective p21 function (andotherwise normal HADH function), can be used in the methods of thepresent invention. For example, a p21 knockout mouse can be used toassess, in vivo, the activity of a candidate p21 modulating agentidentified in one of the in vitro assays described below. p21 knockoutmouse are described in the literature (Umanoff H, et al., Proc Natl AcadSci USA 1995 Feb. 28; 92(5):1709-13). Preferably, the candidate p21modulating agent when administered to a model system with cellsdefective in p21 function, produces a detectable phenotypic change inthe model system indicating that the p21 function is restored, i.e., thecells exhibit normal cell cycle progression.

Modulating Agents

The invention provides methods to identify agents that interact withand/or modulate the function of HADH and/or the p21 pathway. Such agentsare useful in a variety of diagnostic and therapeutic applicationsassociated with the p21 pathway, as well as in further analysis of theHADH protein and its contribution to the p21 pathway. Accordingly, theinvention also provides methods for modulating the p21 pathwaycomprising the step of specifically modulating HADH activity byadministering a HADH-interacting or -modulating agent.

In a preferred embodiment, HADH-modulating agents inhibit or enhanceHADH activity or otherwise affect normal HADH function, includingtranscription, protein expression, protein localization, and cellular orextra-cellular activity. In a further preferred embodiment, thecandidate p21 pathway-modulating agent specifically modulates thefunction of the HADH. The phrases “specific modulating agent”,“specifically modulates”, etc., are used herein to refer to modulatingagents that directly bind to the HADH polypeptide or nucleic acid, andpreferably inhibit, enhance, or otherwise alter, the function of theHADH. The term also encompasses modulating agents that alter theinteraction of the HADH with a binding partner or substrate (e.g. bybinding to a binding partner of an HADH, or to a protein/binding partnercomplex, and inhibiting function).

Preferred HADH-modulating agents include small molecule compounds;HADH-interacting proteins, including antibodies and otherbiotherapeutics; and nucleic acid modulators such as antisense and RNAinhibitors. The modulating agents may be formulated in pharmaceuticalcompositions, for example, as compositions that may comprise otheractive ingredients, as in combination therapy, and/or suitable carriersor excipients. Techniques for formulation and administration of thecompounds may be found in “Remington's Pharmaceutical Sciences” MackPublishing Co., Easton, Pa., 19th edition.

Small Molecule Modulators

Small molecules, are often preferred to modulate function of proteinswith enzymatic function, and/or containing protein interaction domains.Chemical agents, referred to in the art as “small molecule” compoundsare typically organic, non-peptide molecules, having a molecular weightless than 10,000, preferably less than 5,000, more preferably less than1,000, and most preferably less than 500. This class of modulatorsincludes chemically synthesized molecules, for instance, compounds fromcombinatorial chemical libraries. Synthetic compounds may be rationallydesigned or identified based on known or inferred properties of the HADHprotein or may be identified by screening compound libraries.Alternative appropriate modulators of this class are natural products,particularly secondary metabolites from organisms such as plants orfungi, which can also be identified by screening compound libraries forHADH-modulating activity. Methods for generating and obtaining compoundsare well known in the art (Schreiber S L, Science (2000) 151: 1964-1969;Radmann J and Gunther J, Science (2000) 151:1947-1948).

Small molecule modulators identified from screening assays, as describedbelow, can be used as lead compounds from which candidate clinicalcompounds may be designed, optimized, and synthesized. Such clinicalcompounds may have utility in treating pathologies associated with thep21 pathway. The activity of candidate small molecule modulating agentsmay be improved several-fold through iterative secondary functionalvalidation, as further described below, structure determination, andcandidate modulator modification and testing. Additionally, candidateclinical compounds are generated with specific regard to clinical andpharmacological properties. For example, the reagents may be derivatizedand re-screened sing in vitro and in vivo assays to optimize activityand minimize toxicity for pharmaceutical development.

Protein Modulators

Specific HADH-interacting proteins are useful in a variety of diagnosticand therapeutic applications related to the p21 pathway and relateddisorders, as well as in validation assays for other HADH-modulatingagents. In a preferred embodiment, HADH-interacting proteins affectnormal HADH function, including transcription, protein expression,protein localization, and cellular or extra-cellular activity. Inanother embodiment, HADH-interacting proteins are useful in detectingand providing information about the function of HADH proteins, as isrelevant to p21 related disorders, such as cancer (e.g., for diagnosticmeans).

An HADH-interacting protein may be endogenous, i.e. one that naturallyinteracts genetically or biochemically with an HADH, such as a member ofthe HADH pathway that modulates HADH expression, localization, and/oractivity. HADH-modulators include dominant negative forms ofHADH-interacting proteins and of HADH proteins themselves. Yeasttwo-hybrid and variant screens offer preferred methods for identifyingendogenous HADH-interacting proteins (Finley, R. L. et al. (1996) in DNACloning-Expression Systems: A Practical Approach, eds. Glover D. & HamesB. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SFet al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999)3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; andU.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferredmethod for the elucidation of protein complexes (reviewed in, e.g.,Pandley A and Mann M, Nature (2000)405:837-846; Yates J R 3rd, TrendsGenet (2000) 16:5-8).

An HADH-interacting protein may be an exogenous protein, such as anHADH-specific antibody or a T-cell antigen receptor (see, e.g., Harlowand Lane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory; Harlow and Lane (1999) Using antibodies: a laboratorymanual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).HADH antibodies are further discussed below.

In preferred embodiments, an HADH-interacting protein specifically bindsan HADH protein. In alternative preferred embodiments, anHADH-modulating agent binds an HADH substrate, binding partner, orcofactor.

Antibodies

In another embodiment, the protein modulator is an HADH specificantibody agonist or antagonist. The antibodies have therapeutic anddiagnostic utilities, and can be used in screening assays to identifyHADH modulators. The antibodies can also be used in dissecting theportions of the HADH pathway responsible for various cellular responsesand in the general processing and maturation of the HADH.

Antibodies that specifically bind HADH polypeptides can be generatedusing known methods. Preferably the antibody is specific to a mammalianortholog of HADH polypeptide, and more preferably, to human HADH.Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′).sub.2fragments, fragments produced by a FAb expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above. Epitopes of HADH which are particularly antigenic canbe selected, for example, by routine screening of HADH polypeptides forantigenicity or by applying a theoretical method for selecting antigenicregions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci.U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89;Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequenceshown in SEQ ID NOs:4 or 5. Monoclonal antibodies with affinities of 10⁸M⁻¹ preferably 10⁹ M⁻¹ to 10¹⁰ M⁻¹, or stronger can be made by standardprocedures as described (Harlow and Lane, supra; Goding (1986)Monoclonal Antibodies Principles and Practice (2d ed) Academic Press,New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).Antibodies may be generated against crude cell extracts of HADH orsubstantially purified fragments thereof. If HADH fragments are used,they preferably comprise at least 10, and more preferably, at least 20contiguous amino acids of an HADH protein. In a particular embodiment,HADH-specific antigens and/or immunogens are coupled to carrier proteinsthat stimulate the immune response. For example, the subjectpolypeptides are covalently coupled to the keyhole limpet hemocyanin(KLH) carrier, and the conjugate is emulsified in Freund's completeadjuvant, which enhances the immune response. An appropriate immunesystem such as a laboratory rabbit or mouse is immunized according toconventional protocols.

The presence of HADH-specific antibodies is assayed by an appropriateassay such as a solid phase enzyme-linked immunosorbant assay (ELISA)using immobilized corresponding HADH polypeptides. Other assays, such asradioimmunoassays or fluorescent assays might also be used.

Chimeric antibodies specific to HADH polypeptides can be made thatcontain different portions from different animal species. For instance,a human immunoglobulin constant region may be linked to a variableregion of a murine mAb, such that the antibody derives its biologicalactivity from the human antibody, and its binding specificity from themurine fragment. Chimeric antibodies are produced by splicing togethergenes that encode the appropriate regions from each species (Morrison etal., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al.,Nature (1984) 312:604608; Takeda et al., Nature (1985) 31:452-454).Humanized antibodies, which are a form of chimeric antibodies, can begenerated by grafting complementary-determining regions (CDRs) (Carlos,T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies intoa background of human framework regions and constant regions byrecombinant DNA technology (Riechmann L M, et al., 1988 Nature 323:323-327). Humanized antibodies contain −4.0% murine sequences and −90%human sequences, and thus further reduce or eliminate immunogenicity,while retaining the antibody specificities (Co M S, and Queen C. 1991Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun. 10:239-265).Humanized antibodies and methods of their production are well-known inthe art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).

HADH-specific single chain antibodies which are recombinant, singlechain polypeptides formed by linking the heavy and light chain fragmentsof the Fv regions via an amino acid bridge, can be produced by methodsknown in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988)242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988)85:5879-5883; and Ward et al., Nature (1989) 334:544-546).

Other suitable techniques for antibody production involve in vitroexposure of lymphocytes to the antigenic polypeptides or alternativelyto selection of libraries of antibodies in phage or similar vectors(Huse et al., Science (1989) 246:1275-1281). As used herein, T-cellantigen receptors are included within the scope of antibody modulators(Harlow and Lane, 1988, supra).

The polypeptides and antibodies of the present invention may be usedwith or without modification. Frequently, antibodies will be labeled byjoining, either covalently or non-covalently, a substance that providesfor a detectable signal, or that is toxic to cells that express thetargeted protein (Menard S, et al., Int J. Biol Markers (1989)4:131-134). A wide variety of labels and conjugation techniques areknown and ′ are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent moieties, fluorescent emittinglanthanide metals, chemiluminescent moieties, bioluminescent moieties,magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also,recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567).Antibodies to cytoplasmic polypeptides may be delivered and reach theirtargets by conjugation with membrane-penetrating toxin proteins (U.S.Pat. No. 6,086,900).

When used therapeutically in a patient, the antibodies of the subjectinvention are typically administered parenterally, when possible at thetarget site, or intravenously. The therapeutically effective dose anddosage regimen is determined by clinical studies. Typically, the amountof antibody administered is in the range of about 0.1 mg/kg—to about 10mg/kg of patient weight. For parenteral administration, the antibodiesare formulated in a unit dosage injectable form (e.g., solution,suspension, emulsion) in association with a pharmaceutically acceptablevehicle. Such vehicles are inherently nontoxic and non-therapeutic.Examples are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils, ethyloleate, or liposome carriers may also be used. The vehicle may containminor amounts of additives, such as buffers and preservatives, whichenhance isotonicity and chemical stability or otherwise enhancetherapeutic potential. The antibodies' concentrations in such vehiclesare typically in the range of about 1 mg/ml to about 10 mg/ml.

Immunotherapeutic methods are further described in the literature (U.S.Pat. No. 5,859,206; WO0073469).

Nucleic Acid Modulators

Other preferred HADH-modulating agents comprise nucleic acid molecules,such as antisense oligomers or double stranded RNA (dsRNA), whichgenerally inhibit HADH activity. Preferred nucleic acid modulatorsinterfere with the function of the HADH nucleic acid such as DNAreplication, transcription, translocation of the HADH RNA to the site ofprotein translation, translation of protein from the HADH RNA, splicingof the HADH RNA to yield one or more mRNA species, or catalytic activitywhich may be engaged in or facilitated by the HADH RNA.

In one embodiment, the antisense oligomer is an oligonucleotide that issufficiently complementary to an HADH mRNA to bind to and preventtranslation, preferably by binding to the 5′ untranslated region.HADH-specific antisense oligonucleotides, preferably range from at least6 to about 200 nucleotides. In some embodiments the oligonucleotide ispreferably at least 10, 15, or 20 nucleotides in length. In otherembodiments, the oligonucleotide is preferably less than 50, 40, or 30nucleotides in length. The oligonucleotide can be DNA or RNA or achimeric mixture or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides,agents that facilitate transport across the cell membrane,hybridization-triggered cleavage agents, and intercalating agents.

In another embodiment, the antisense oligomer is a phosphothioatemorpholino oligomer (PMO). PMOs are assembled from four differentmorpholino subunits, each of which contain one of four genetic bases (A,C, G, or T) linked to a six-membered morpholine ring. Polymers of thesesubunits are joined by non-ionic phosphodiamidate intersubunit linkages.Details of how to make and use PMOs and other antisense oligomers arewell known in the art (e.g. see WO99/18193; Probst J C, AntisenseOligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281;Summerton J, and Weller D. 1997 Antisense Nucleic Acid DrugDev.:7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).

Alternative preferred HADH nucleic acid modulators are double-strandedRNA species mediating RNA interference (RNAi). RNAi is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by double-stranded RNA (dsRNA) that is homologous insequence to the silenced gene. Methods relating to the use of RNAi tosilence genes in C. elegans, Drosophila, plants, and humans are known inthe art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet.15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15,485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119(2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. etal., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404,293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., etal., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619; Elbashir S M,et al., 2001 Nature 411:494-498).

Nucleic acid modulators are commonly used as research reagents,diagnostics, and therapeutics. For example, antisense oligonucleotides,which are able to inhibit gene expression with exquisite specificity,are often used to elucidate the function of particular genes (see, forexample, U.S. Pat. No. 6,165,790). Nucleic acid modulators are alsoused, for example, to distinguish between functions of various membersof a biological pathway. For example, antisense oligomers have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man and have been demonstrated in numerous clinical trialsto be safe and effective (Milligan J F, et al, Current Concepts inAntisense Drug Design, J Med Chem. (1993) 36:1923-1937; Tonkinson J L etal., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents,Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of theinvention, an HADH-specific nucleic acid modulator is used in an assayto further elucidate the role of the HADH in the p21 pathway, and/or itsrelationship to other members of the pathway. In another aspect of theinvention, an HADH-specific antisense oligomer is used as a therapeuticagent for treatment of p21-related disease states.

Assay Systems

The invention provides assay systems and screening methods foridentifying specific modulators of HADH activity. As used herein, an“assay system” encompasses all the components required for performingand analyzing results of an assay that detects and/or measures aparticular event. In general, primary assays are used to identify orconfirm a modulator's specific biochemical or molecular effect withrespect to the HADH nucleic acid or protein. In general, secondaryassays further assess the activity of a HADH modulating agent identifiedby a primary assay and may confirm that the modulating agent affectsHADH in a manner relevant to the p21 pathway. In some cases, HADHmodulators will be directly tested in a secondary assay.

In a preferred embodiment, the screening method comprises contacting asuitable assay system comprising an HADH polypeptide with a candidateagent under conditions whereby, but for the presence of the agent, thesystem provides a reference activity (e.g. binding activity), which isbased on the particular molecular event the screening method detects. Astatistically significant difference between the agent-biased activityand the reference activity indicates that the candidate agent modulatesHADH activity, and hence the p21 pathway.

Primary Assays

The type of modulator tested generally determines the type of primaryassay.

Primary Assays for Small Molecule Modulators

For small molecule modulators, screening assays are used to identifycandidate modulators. Screening assays may be cell-based or may use acell-free system that recreates or retains the relevant biochemicalreaction of the target protein (reviewed in Sittampalam G S et al., CurrOpin Chem Biol (1997) 1:384-91 and accompanying references). As usedherein the term “cell-based” refers to assays using live cells, deadcells, or a particular cellular fraction, such as a membrane,endoplasmic reticulum, or mitochondrial fraction. The term “cell free”encompasses assays using substantially purified protein (eitherendogenous or recombinantly produced), partially purified or crudecellular extracts. Screening assays may detect a variety of molecularevents, including protein-DNA interactions, protein-protein interactions(e.g., receptor-ligand binding), transcriptional activity (e.g., using areporter gene), enzymatic activity (e.g., via a property of thesubstrate), activity of second messengers, immunogenicty and changes incellular morphology or-other cellular characteristics. Appropriatescreening assays may use a wide range of detection methods includingfluorescent, radioactive, colorimetric, spectrophotometric, andamperometric methods, to provide a read-out for the particular molecularevent detected.

Cell-based screening assays usually require systems for recombinantexpression of HADH and any auxiliary proteins demanded by the particularassay. Appropriate methods for generating recombinant proteins producesufficient quantities of proteins that retain their relevant biologicalactivities and are of sufficient purity to optimize activity and assureassay reproducibility. Yeast two-hybrid and variant screens, and massspectrometry provide preferred methods for determining protein-proteininteractions and elucidation of protein complexes. In certainapplications, when HADH-interacting proteins are used in screens toidentify small molecule modulators, the binding specificity of theinteracting protein to the HADH protein may be assayed by various knownmethods such as substrate processing (e.g. ability of the candidate HADHspecific binding agents to function as negative effectors inHADH-expressing cells), binding equilibrium constants (usually at leastabout 10⁷M⁻¹, preferably at least about 10⁸ W^(I), more preferably atleast about 10⁹ M⁻¹), and immunogenicity (e.g. ability to elicit HADHspecific antibody in a heterologous host such as a mouse, rat, goat orrabbit). For enzymes and receptors, binding may be assayed by,respectively, substrate and ligand processing.

The screening assay may measure a candidate agent's ability tospecifically bind to or modulate activity of a HADH polypeptide, afusion protein thereof, or to cells or membranes bearing the polypeptideor fusion protein. The HADH polypeptide can be full length or a fragmentthereof that retains functional HADH activity. The HADH polypeptide maybe fused to another polypeptide, such as a peptide tag for detection oranchoring, or to another tag. The HADH polypeptide is preferably humanHADH, or is an ortholog or derivative thereof as described above. In apreferred embodiment, the screening assay detects candidate agent-basedmodulation of HADH interaction with a binding target, such as anendogenous or exogenous protein or other substrate that hasHADH-specific binding activity, and can be used to assess normal HADHgene function.

Suitable assay formats that may be adapted to screen for HADH modulatorsare known in the art. Preferred screening assays are high throughput orultra high throughput and thus provide automated, cost-effective meansof screening compound libraries for lead compounds (Fernandes P B, CurrOpin Chem Biol (1998) 2:597-603; Sundberg S A, Curr Opin Biotechnol2000, 11:47-53). In one preferred embodiment, screening assays usesfluorescence technologies, including fluorescence polarization,time-resolved fluorescence, and fluorescence resonance energy transfer.These systems offer means to monitor protein-protein or DNA-proteininteractions in which the intensity of the signal emitted fromdye-labeled molecules depends upon their interactions with partnermolecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; FernandesPB, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000)4:445-451).

A variety of suitable assay systems may be used to identify candidateHADH and p21 pathway modulators (e.g. U.S. Pat. Nos. 5,550,019 and6,133,437 (apoptosis assays), among others). Specific preferred assaysare described in more detail below.

Hydrolase assays. Hydrolases catalyze the hydrolysis of a substrate suchas esterases, lipases, peptidases, nucleotidases, and phosphatases,among others. Enzyme activity assays may be used to measure hydrolaseactivity. The activity of the enzyme is determined in presence of excesssubstrate, by spectrophotometrically measuring the rate of appearance ofreaction products. High throughput arrays and assays for hydrolases areknown to those skilled in the art (Park C B and Clark D S (2002) BiotechBioeng 78:229-235).

Apoptosis assays. Assays for apoptosis may be performed by terminaldeoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick endlabeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNAfragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature371, 346), by following the incorporation of fluorescein-dUTP (Yoneharaet al., 1989, J. Exp. Med. 169, 1747). Apoptosis may further be assayedby acridine orange staining of tissue culture cells (Lucas, R., et al.,1998, Blood 15:4730-41). An apoptosis assay system may comprise a cellthat expresses an HADH, and that optionally has defective p21 function(e.g. p21 is over-expressed or under-expressed relative to wild-typecells). A test agent can be added to the apoptosis assay system andchanges in induction of apoptosis relative to controls where no testagent is added, identify candidate p21 modulating agents. In someembodiments of the invention, an apoptosis assay may be used as asecondary assay to test a candidate p21 modulating agents that isinitially identified using a cell-free assay system. An apoptosis assaymay also be used to test whether HADH function plays a direct role inapoptosis. For example, an apoptosis assay may be performed on cellsthat over- or under-express HADH relative to wild type cells.Differences in apoptotic response compared to wild type cells suggeststhat the HADH plays a direct role in the apoptotic response. Apoptosisassays are described further in U.S. Pat. No. 6,133,437.

Cell proliferation and cell cycle assays. Cell proliferation may beassayed via bromodeoxyuridine (BRDU) incorporation. This assayidentifies a cell population undergoing DNA synthesis by incorporationof BRDU into newly-synthesized DNA. Newly-synthesized DNA may then bedetected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J.Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or byother means.

Cell Proliferation may also be examined using [³H]-thymidineincorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995,J. Biol. Chem. 270:18367-73). This assay allows for quantitativecharacterization of S-phase DNA syntheses. In this assay, cellssynthesizing DNA will incorporate [³H]-thymidine into newly synthesizedDNA. Incorporation can then be measured by standard techniques such asby counting of radioisotope in a scintillation counter (e.g., Beckman LS3800 Liquid Scintillation Counter).

Cell proliferation may also be assayed by colony formation in soft agar(Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). Forexample, cells transformed with HADH are seeded in soft agar plates, andcolonies are measured and counted after two weeks incubation.Involvement of a gene in the cell cycle may be assayed by flow cytometry(Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med49:237-55). Cells transfected with an HADH may be stained with propidiumiodide and evaluated in a flow cytometer (available from BectonDickinson).

Accordingly, a cell proliferation or cell cycle assay system maycomprise a cell that expresses an HADH, and that optionally hasdefective p21 function (e.g. p21 is over-expressed or under-expressedrelative to wild-type cells). A test agent can be added to the assaysystem and changes in cell proliferation or cell cycle relative tocontrols where no test agent is added, identify candidate p21 modulatingagents. In some embodiments of the invention, the cell proliferation orcell cycle assay may be used as a secondary assay to test a candidatep21 modulating agents that is initially identified using another assaysystem such as a cell-free kinase assay system. A cell proliferationassay may also be used to test whether HADH function plays a direct rolein cell proliferation or cell cycle. For example, a cell proliferationor cell cycle assay may be performed on cells that over- orunder-express HADH relative to wild type cells. Differences inproliferation or cell cycle compared to wild type cells suggests thatthe HADH plays a direct role in cell proliferation or cell cycle.

Angiogenesis. Angiogenesis may be assayed using various humanendothelial cell systems, such as umbilical vein, coronary artery, ordermal cells. Suitable assays include Alamar Blue based assays(available from Biosource International) to measure proliferation;migration assays using fluorescent molecules, such as the use of BectonDickinson Falcon HTS FluoroBlock cell culture inserts to measuremigration of cells through membranes in presence or absence ofangiogenesis enhancer or suppressors; and tubule formation assays basedon the formation of tubular structures by endothelial cells on Matrigel®(Becton Dickinson). Accordingly, an angiogenesis assay system maycomprise a cell that expresses an HADH, and that optionally hasdefective p21 function (e.g. p21 is over-expressed or under-expressedrelative to wild-type cells). A test agent can be added to theangiogenesis assay system and changes in angiogenesis relative tocontrols where no test agent is added, identify candidate p21 modulatingagents. In some embodiments of the invention, the angiogenesis assay maybe used as a secondary assay to test a candidate p21 modulating agentsthat is initially identified using another assay system. An angiogenesisassay may also be used to test whether HADH function plays a direct rolein cell proliferation. For example, an angiogenesis assay may beperformed on cells that over- or under-express HADH relative to wildtype cells. Differences in angiogenesis compared to wild type cellssuggests that the HADH plays a direct role in angiogenesis.

Hypoxic induction. The alpha subunit of the transcription factor,hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cellsfollowing exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1stimulates the expression of genes known to be important in tumour cellsurvival, such as those encoding glyolytic enzymes and VEGF. Inductionof such genes by hypoxic conditions may be assayed by growing cellstransfected with HADH in hypoxic conditions (such as with 0.1% 02, 5%CO2, and balance N2, generated in a Napco 7001 incubator (PrecisionScientific)) and normoxic conditions, followed by assessment of geneactivity or expression by Taqman®. For example, a hypoxic inductionassay system may comprise a cell that expresses an HADH, and thatoptionally has a mutated p21 (e.g. p21 is over-expressed orunder-expressed relative to wild-type cells). A test agent can be addedto the hypoxic induction assay system and changes in hypoxic responserelative to controls where no test agent is added, identify candidatep21 modulating agents. In some embodiments of the invention, the hypoxicinduction assay may be used as a secondary assay to test a candidate p21modulating agents that is initially identified using another assaysystem. A hypoxic induction assay may also be used to test whether HADHfunction plays a direct role in the hypoxic response. For example, ahypoxic induction assay may be performed on cells that over- orunder-express HADH relative to wild type cells. Differences in hypoxicresponse compared to wild type cells suggests that the HADH plays adirect role in hypoxic induction.

Cell adhesion. Cell adhesion assays measure adhesion of cells topurified adhesion proteins, or adhesion of cells to each other, inpresence or absence of candidate modulating agents. Cell-proteinadhesion assays measure the ability of agents to modulate the adhesionof cells to purified proteins. For example, recombinant proteins areproduced, diluted to 2.5 g/mL in PBS, and used to coat the wells of amicrotiter plate. The wells used for negative control are not coated.Coated wells are then washed, blocked with 1% BSA, and washed again.Compounds are diluted to 2× final test concentration and added to theblocked, coated wells. Cells are then added to the wells, and theunbound cells are washed off. Retained cells are labeled directly on theplate by adding a membrane-permeable fluorescent dye, such ascalcein-AM, and the signal is quantified in a fluorescent microplatereader.

Cell-cell adhesion assays measure the ability of agents to modulatebinding of cell adhesion proteins with their native ligands. Theseassays use cells that, naturally or recombinantly express the adhesionprotein of choice. In an exemplary assay, cells expressing the celladhesion protein are plated in wells of a multiwell plate. Cellsexpressing the ligand are labeled with a membrane-permeable fluorescentdye, such as BCECF, and allowed to adhere to the monolayers in thepresence of candidate agents. Unbound cells are washed off, and boundcells are detected using a fluorescence plate reader.

High-throughput cell adhesion assays have also been described. In onesuch assay, small molecule ligands and peptides are bound to the surfaceof microscope slides using a microarray spotter, intact cells are thencontacted with the slides, and unbound cells are washed off. In thisassay, not only the binding specificity of the peptides and modulatorsagainst cell lines are determined, but also the functional cellsignaling of attached cells using immunofluorescence techniques in situon the microchip is measured (Falsey J R et al., Bioconjug Chem. 2001May-June; 12(3):346-53).

Primary Assays for Antibody Modulators

For antibody modulators, appropriate primary assays test is a bindingassay that tests the antibody's affinity to and specificity for the HADHprotein. Methods for testing antibody affinity and specificity are wellknown in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linkedimmunosorbant assay (ELISA) is a preferred method for detectingHADH-specific antibodies; others include FACS assays, radioimmunoassays,and fluorescent assays.

Primary Assays for Nucleic Acid Modulators

For nucleic acid modulators, primary assays may test the ability of thenucleic acid modulator to inhibit or enhance HADH gene expression,preferably mRNA expression. In general, expression analysis comprisescomparing HADH expression in like populations of cells (e.g., two poolsof cells that endogenously or recombinantly express HADH) in thepresence and absence of the nucleic acid modulator. Methods foranalyzing mRNA and protein expression are well known in the art. Forinstance, Northern blotting, slot blotting, ribonuclease protection,quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), ormicroarray analysis may be used to confirm that HADH mRNA expression isreduced in cells treated with the nucleic acid modulator (e.g., CurrentProtocols in Molecular Biology (1994) Ausubel F M et al., eds., JohnWiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999)26:112125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm DH andGuiseppi-Elie, A. Curr Opin Biotechnol 2001, 12:41-47). Proteinexpression may also be monitored. Proteins are most commonly detectedwith specific antibodies or antisera directed against either the HADHprotein or specific peptides. A variety of means including Westernblotting, ELISA, or in situ detection, are available (Harlow E and LaneD, 1988 and 1999, supra).

Secondary Assays

Secondary assays may be used to further assess the activity ofHADH-modulating agent identified by any of the above methods to confirmthat the modulating agent affects HADH in a manner relevant to the p21pathway. As used herein, HADHmodulating agents encompass candidateclinical compounds or other agents derived from previously identifiedmodulating agent. Secondary assays can also be used to test the activityof a modulating agent on a particular genetic or biochemical pathway orto test the specificity of the modulating agent's interaction with HADH.

Secondary assays generally compare like populations of cells or animals(e.g., two pools of cells or animals that endogenously or recombinantlyexpress HADH) in the presence and absence of the candidate modulator. Ingeneral, such assays test whether treatment of cells or animals with acandidate HADH-modulating agent results in changes in the p21 pathway incomparison to untreated (or mock- or placebo-treated) cells or animals.Certain assays use “sensitized genetic backgrounds”, which, as usedherein, describe cells or animals engineered for altered expression ofgenes in the p21 or interacting pathways.

Cell-Based Assays

Cell based assays may use a variety of a cell line known to havedefective p21 function such as HCT116 colon cancer cells available fromAmerican Type Culture Collection (ATCC), Manassas, Va.). Cell basedassays may detect endogenous p21 pathway activity or may rely onrecombinant expression of p21 pathway components. Any of theaforementioned assays may be used in this cell-based format. Candidatemodulators are typically added to the cell media but may also beinjected into cells or delivered by any other efficacious means.

Animal Assays

A variety of non-human animal models of normal or defective p21 pathwaymay be used to test candidate HADH modulators. Models for defective p21pathway typically use genetically modified animals that have beenengineered to mis-express (e.g., over-express or lack expression in)genes involved in the p21 pathway. Assays generally require systemicdelivery of the candidate modulators, such as by oral administration,injection, etc.

In a preferred embodiment, p21 pathway activity is assessed bymonitoring neovascularization and angiogenesis. Animal models withdefective and normal p21 are used to test the candidate modulator'saffect on HADH in Matrigel® assays. Matrigel® is an extract of basementmembrane proteins, and is composed primarily of laminin, collagen IV,and heparin sulfate proteoglycan. It is provided as a sterile liquid at4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixedwith various angiogenic agents, such as bFGF and VEGF, or with humantumor cells which over-express the HADH. The mixture is then injectedsubcutaneously(SC) into female athymic nude mice (Taconic, Germantown,N.Y.) to support an intense vascular response. Mice with Matrigel®pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous(IV) routes with the candidate modulator. Mice are euthanized 5-12 dayspost-injection, and the Matrigel® pellet is harvested for hemoglobinanalysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel isfound to correlate the degree of neovascularization in the gel.

In another preferred embodiment, the effect of the candidate modulatoron HADH is assessed via tumorigenicity assays. In one example, xenografthuman tumors are implanted SC into female athymic mice, 6-7 week old, assingle cell suspensions either from a pre-existing tumor or from invitro culture. The tumors which express the HADH endogenously areinjected in the flank, 1×10⁵ to 1×10⁷ cells per mouse in a volume of 100lit using a 27 gauge needle. Mice are then ear tagged and tumors aremeasured twice weekly. Candidate modulator treatment is initiated on theday the mean tumor weight reaches 100 mg. Candidate modulator isdelivered IV, SC, IP, or PO by bolus administration. Depending upon thepharmacokinetics of each unique candidate modulator, dosing can beperformed multiple times per day. The tumor weight is assessed bymeasuring perpendicular diameters with a caliper and calculated bymultiplying the measurements of diameters in two dimensions. At the endof the experiment, the excised tumors maybe utilized for biomarkeridentification or further analyses. For immunohistochemistry staining,xenograft tumors are fixed in 4% paraformaldehyde, 0.1M phosphate, pH7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidlyfrozen in isopentane cooled with liquid nitrogen.

Diagnostic and Therapeutic Uses

Specific HADH-modulating agents are useful in a variety of diagnosticand therapeutic applications where disease or disease prognosis isrelated to defects in the p21 pathway, such as angiogenic, apoptotic, orcell proliferation disorders. Accordingly, the invention also providesmethods for modulating the p21 pathway in a cell, preferably a cellpre-determined to have defective p21 function, comprising the step ofadministering an agent to the cell that specifically modulates HADHactivity. Preferably, the modulating agent produces a detectablephenotypic change in the cell indicating that the p21 function isrestored, i.e., for example, the cell undergoes normal proliferation orprogression through the cell cycle.

The discovery that HADH is implicated in p21 pathway provides for avariety of methods that can be employed for the diagnostic andprognostic evaluation of diseases and disorders involving defects in thep21 pathway and for the identification of subjects having apredisposition to such diseases and disorders.

Various expression analysis methods can be used to diagnose whether HADHexpression occurs in a particular sample, including Northern blotting,slot blotting, ribonuclease protection, quantitative RT-PCR, andmicroarray analysis. (e.g., Current Protocols in Molecular Biology(1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4;Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P,Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol2001, 12:41-47). Tissues having a disease or disorder implicatingdefective p21 signaling that express an HADH, are identified as amenableto treatment with an HADH modulating agent. In a preferred application,the p21 defective tissue overexpresses an HADH relative to normaltissue. For example, a Northern blot analysis of mRNA from tumor andnormal cell lines, or from tumor and matching normal tissue samples fromthe same patient, using full or partial HADH cDNA sequences as probes,can determine whether particular tumors express or overexpress HADH.Alternatively, the TaqMan® is used for quantitative RT-PCR analysis ofHADH expression in cell lines, normal tissues and tumor samples (PEApplied Biosystems).

Various other diagnostic methods may be performed, for example,utilizing reagents such as the HADH oligonucleotides, and antibodiesdirected against an HADH, as described above for: (1) the detection ofthe presence of HADH gene mutations, or the detection of either over- orunder-expression of HADH mRNA relative to the non-disorder state; (2)the detection of either an over- or an under-abundance of HADH geneproduct relative to the non-disorder state; and (3) the detection ofperturbations or abnormalities in the signal transduction pathwaymediated by HADH.

Thus, in a specific embodiment, the invention is drawn to a method fordiagnosing a disease in a patient, the method comprising: a) obtaining abiological sample from the patient; b) contacting the sample with aprobe for HADH expression; c) comparing results from step (b) with acontrol; and d) determining whether step (c) indicates a likelihood ofdisease. Preferably, the disease is cancer, most preferably colon orovarian cancer. The probe may be either DNA or protein, including anantibody.

EXAMPLES

The following experimental section and examples are offered by way ofillustration and not by way of limitation.

I. Drosophila p21 screen

An overexpression screen was carried out in Drosophila to identify genesthat interact with the cyclin dependent kinase inhibitor, p21 (Bourne HR, et al., Nature (1990) 348(6297):125-132; Marshall C J, Trends Genet(1991) 7(3):91-95). Expression of the p21 gene in the eye causesdeterioration of normal eye morphology. Modifiers of the eye phenotypewere identified as members of the p21 pathway. CG 15771 was a suppressorof the small eye defect.

BLAST analysis (Altschul et al., supra) was employed to identify Targetsfrom Drosophila modifiers. For example, are preventative sequence fromHADH (GI# 4902680, SEQ ID NO:4) shares 34% amino acid identity with theDrosophila CG 15771.

Various domains, signals, and functional subunits in proteins wereanalyzed using the PSORT (Nakai K., and Horton P., Trends Biochem Sci,1999, 24:34-6; Kenta Nakai, Protein sorting signals and prediction ofsubcellular localization, Adv. Protein Chem. 54, 277-344 (2000)), PFAM(Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2;http://pfam.wustl.edu), SMART (Ponting C P, et al., SMART:identification and annotation of domains from signaling andextracellular protein sequences. Nucleic Acids Res. 1999 Jan. 1;27(1):229-32), TM-HMM (Erik L. L. Sonnhammer, Gunnar von Heijne, andAnders Krogh: A hidden Markov model for predicting transmembrane helicesin protein sequences. In Proc. of Sixth Int. Conf. on IntelligentSystems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn,F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAIPress, 1998), and dust (Remm M, and Sonnhammer E. Classification oftransmembrane protein families in the Caenorhabditis elegans genome andidentification of human orthologs. Genome Res. 2000 November;10(11):1679-89) programs. For example, the hydrolase domain of HADH fromGI# 4902680 (SEQ ID NO:4) is located at approximately amino acidresidues 9-212 (PFAM 00702).

Detailed functional analysis of SEQ ID NO:5 indicated that HADH appearsto be a member of the family of the HADH aspartyl-phosphate utilizingphosphohydrolases/phosphotransferases. We used Threading algorithm(Proceryon, N.Y.) to identify structure family relationship of HADH.Threading alignment identified several key residues for members of thisfamily: D12, T16, T131, N132, K164, D189, T193, D194. In SEQ ID NO:5,D12 is highly conserved, and is the site of phosphorylation; T16 issomewhat variable, and appears to impact rate of autohydrolysis of theD-phosphate; T131 likely involved in coordination of phosphate; K164likely involved in activating the water molecule that hydrolyzes theacyl intermediate, and/or involved in coordination of oxygen inacyl-phosphate/stabilization of phosphorylated state; D189,D194 likelycoordinate Mg or other metal cation. (Mg or other metal cations are notconserved in epoxide hydrolases and dehalogenases, but are conserved andrequired for the activity of phosphohydrolases/phosphotransferases).

II. High-Throughput In Vitro Fluorescence Polarization Assay

Fluorescently-labeled HADH peptide/substrate are added to each well of a96-well microtiter plate, along with a test agent in a test buffer (10mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes influorescence polarization, determined by using a Fluorolite FPM-2Fluorescence Polarization Microtiter System (Dynatech Laboratories,Inc), relative to control values indicates the test compound is acandidate modifier of HADH activity.

High-Throughput In Vitro Binding Assay.

³³P-labeled HADH peptide is added in an assay buffer (100 mM KC1, 20 mMHEPES pH 7.6, 1 mM MgC1₂, 1% glycerol, 0.5% NP-40, 50 mMbeta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors)along with a test agent to the wells of a Neutralite-avidin coated assayplate and incubated at 25° C. for 1 hour. Biotinylated substrate is thenadded to each well and incubated for 1 hour. Reactions are stopped bywashing with PBS, and counted in a scintillation counter. Test agentsthat cause a difference in activity relative to control without testagent are identified as candidate p21 modulating agents.

Immunoprecipitations and Immunoblotting

For coprecipitation of transfected proteins, 3×10⁶ appropriaterecombinant cells containing the HADH proteins are plated on 10-cmdishes and transfected on the following day with expression constructs.The total amount of DNA is kept constant in each transfection by addingempty vector. After 24 h, cells are collected, washed once withphosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysisbuffer containing 50 mM Hepes, pH 7.9, 250 mM NaC1, 20mM-glycerophosphate, 1 mM sodium orthovanadate, 5 mM p-nitrophenylphosphate, 2 mM dithiothreitol, protease inhibitors (complete, RocheMolecular Biochemicals), and 1% Nonidet P-40. Cellular debris is removedby centrifugation twice at 15,000×g for 15 min. The cell lysate isincubated with 25 μl of M2 beads (Sigma) for 2 h at 4° C. with gentlerocking.

After extensive washing with lysis buffer, proteins bound to the beadsare solubilized by boiling in SDS sample buffer, fractionated bySDS-polyacrylamide gel electrophoresis, transferred to polyvinylidenedifluoride membrane and blotted with the indicated antibodies. Thereactive bands are visualized with horseradish peroxidase coupled to theappropriate secondary antibodies and the enhanced chemiluminescence(ECL) Western blotting detection system (Amersham Pharmacia Biotech).

Expression Analysis

All cell lines used in the following experiments are NCI (NationalCancer Institute) lines, and are available from ATCC (American TypeCulture Collection, Mnassas, Va. 20110-2209). Normal and tumor tissueswere obtained from Impath, UC Davis, Clontech, Stratagene, and Ambion.

TaqMan analysis was used to assess expression levels of the disclosedgenes in various samples.

RNA was extracted from each tissue sample using Qiagen (Valencia,Calif.) RNeasy kits, following manufacturer's protocols, to a finalconcentration of 50 ng/gl. Single stranded cDNA was then synthesized byreverse transcribing the RNA samples using random hexamers and 500 ng oftotal RNA per reaction, following protocol 430-4965 of AppliedBiosystems (Foster City, Calif., http://www.appliedbiOsystems.com/).

Primers for expression analysis using TaqMan assay (Applied Biosystems,Foster City, Calif.) were prepared according to the TaqMan protocols,and the following criteria:

-   -   a) primer pairs were designed to span introns to eliminate        genomic contamination, and    -   b) each primer pair produced only one product.

Taqman reactions were carried out following manufacturer's protocols, in25 R1 total volume for 96-well plates and 10 total volume for 384-wellplates, using 300 nM primer and 250 nM probe, and approximately 25 ng ofcDNA. The standard curve for result analysis was prepared using auniversal pool of human cDNA samples, which is a mixture of cDNAs from awide variety of tissues so that the chance that a target will be presentin appreciable amounts is good. The raw data were normalized using 18SrRNA (universally expressed in all tissues and cells).

For each expression analysis, tumor tissue samples were compared withmatched normal tissues from the same patient. A gene was consideredoverexpressed in a tumor when the level of expression of the gene was 2fold or higher in the tumor compared with its matched normal sample. Incases where normal tissue was not available, a universal pool of cDNAsamples was used instead. In these cases, a gene was consideredoverexpressed in a tumor sample when the difference of expression levelsbetween a tumor sample and the average of all normal samples from thesame tissue type was greater than 2 times the standard deviation of allnormal samples (i.e., Tumor an aerage (all normal samples)>2×STDEV (allnormal samples)).

HADH GI#11968366 (SEQ ID NO:1) was overexpressed in 10 of 30 matchedcolon tumors, and 2 of 7 matched ovarian tumors. A modulator identifiedby an assay described herein can be further validated for therapeuticeffect by administration to a tumor in which the gene is overexpressed.A decrease in tumor growth confirms therapeutic utility of themodulator. Prior to treating a patient with the modulator, thelikelihood that the patient will respond to treatment can be diagnosedby obtaining a tumor sample from the patient, and assaying forexpression of the gene targeted by the modulator. The expression datafor the gene(s) can also be used as a diagnostic marker for diseaseprogression. The assay can be performed by expression analysis asdescribed above, by antibody directed to the gene target, or by anyother available detection method.

1. A method of identifying a candidate p21 pathway modulating agent,said method comprising the steps of: (a) providing an assay systemcomprising a purified HADH polypeptide or nucleic acid or a functionallyactive fragment or derivative thereof; (b) contacting the assay systemwith a test agent under conditions whereby, but for the presence of thetest agent, the system provides a reference activity; and (c) detectinga test agent-biased activity of the assay system, wherein a differencebetween the test agent-biased activity and the reference activityidentifies the test agent as a candidate p21 pathway modulating agent.2. The method of claim 1 wherein the assay system comprises culturedcells that express the HADH polypeptide.
 3. The method of claim 2wherein the cultured cells additionally have defective p21 function. 4.The method of claim 1 wherein the assay system includes a screeningassay comprising a HADH polypeptide, and the candidate test agent is asmall molecule modulator.
 5. The method of claim 4 wherein the assay isa binding assay.
 6. The method of claim 1 wherein the assay system isselected from the group consisting of an apoptosis assay system, a cellproliferation assay system, an angiogenesis assay system, and a hypoxicinduction assay system.
 7. The method of claim 1 wherein the assaysystem includes a binding assay comprising a HADH polypeptide and thecandidate test agent is an antibody.
 8. The method of claim 1 whereinthe assay system includes an expression assay comprising a HADH nucleicacid and the candidate test agent is a nucleic acid modulator.
 9. Themethod of claim 8 wherein the nucleic acid modulator is an antisenseoligomer.
 10. The method of claim 8 wherein the nucleic acid modulatoris a PMO.
 11. The method of claim 1 additionally comprising: (d)administering the candidate p21 pathway modulating agent identified in(c) to a model system comprising cells defective in p21 function and,detecting a phenotypic change in the model system that indicates thatthe p21 function is restored.
 12. The method of claim 11 wherein themodel system is a mouse model with defective p21 function. 13.(canceled)
 14. (canceled)
 15. (canceled)
 16. The method of claim 1,comprising the additional steps of: (d) providing a secondary assaysystem comprising cultured cells or a nonhuman animal expressing HADH,contacting the secondary assay system with the test agent of (b) or anagent derived therefrom under conditions whereby, but for the presenceof the test agent or agent derived therefrom, the system provides areference activity; and (e) detecting an agent-biased activity of thesecond assay system, wherein a difference between the agent-biasedactivity and the reference activity of the second assay system confirmsthe test agent or agent derived therefrom as a candidate p21 pathwaymodulating agent, and wherein the second assay detects an agent-biasedchange in the p21 pathway.
 17. The method of claim 16 wherein thesecondary assay system comprises cultured cells.
 18. The method of claim16 wherein the secondary assay system comprises a nonhuman animal 19.The method of claim 18 wherein the non-human animal mis-expresses a p21pathway gene.
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. A methodfor diagnosing a disease in a patient comprising: (a) obtaining abiological sample from the patient; (b) contacting the sample with aprobe for HADH expression; (c) comparing results from step (b) with acontrol; (d) determining whether step (c) indicates a likelihood ofdisease.
 24. The method of claim 23 wherein said disease is cancer. 25.The method according to claim 24, wherein said cancer is colon orovarian cancer.